Purification and properties of monomeric (G1) forms of acetylcholinesterase secreted by Nippostrongylus brasiliensis.
Acetylcholinesterase (AChE) activity secreted by Nippostrongylus brasiliensis was resolved by sucrose density centrifugation and gel permeation chromatography in single peaks estimated at 4.3 S and 60-85 kDa, respectively. Sedimentation was unaffected by the inclusion of detergent. AChE was purified by affinity chromatography on 9-[Nbeta-(epsilon-aminocaproyl)-beta-aminopropylamino]-acridinium bromide hydrobromide-coupled sepharose 4B. Three forms of the enzyme (A, B and C) were distinguished by non-denaturating polyacrylamide gel electrophoresis, and displayed apparent masses of 74, 69 and 71 kDa respectively when resolved by SDS-PAGE. All three isoforms showed a preference for acetylthiocholine (ASCh) as substrate. They were highly sensitive to inhibition by the AChE-specific inhibitor bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with inhibitor concentration reducing initial activity by 50% (IC50) between 0.1 and 0.8 microM, but activity was unaffected by tetramonoisopropylpyrophosphortetramide (iso-OMPA) at concentrations up to 10 mM. We conclude that the secreted enzymes are authentic AChEs of hydrophilic monomeric (G1) form and broadly similar properties, but which can be distinguished by molecular mass, inhibitor sensitivities and the degree of excess substrate inhibition.
Grigg
M E
ME
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
Tang
L
L
Hussein
A S
AS
Selkirk
M E
ME
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000122:Acetylthiocholine / Q000378:metabolism; D000818:Animals; D002499:Centrifugation, Density Gradient; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D009559:Nippostrongylus / Q000201:enzymology* / Q000302:isolation & purification; D013379:Substrate Specificity; D013770:Tetraisopropylpyrophosphamide / Q000494:pharmacology
D002800:Cholinesterase Inhibitors; D000122:Acetylthiocholine; D013770:Tetraisopropylpyrophosphamide; D000110:Acetylcholinesterase